Autism Spectrum Disorders (ASDs) affect 1/110 children in the United States, but physicians do not yet have an effective laboratory test which can confirm the clinical diagnosis. This proposal aims to develop a quick and reliable metabolic array to identify individuals with ASD. Using Phenotype Microarray plates obtained from Biolog (Hayward, CA), we have been able to show that Nicotinamide Adenine Dinucleotide, reduced form (NADH) production in the presence of tryptophan as only energy source in lymphoblastoid cells was consistently reduced in 54/56 (96.4%) patients with ASD when compared to 40 controls. The first aim of the proposed project is to validate our preliminary findings using a cohort of 50 additional patients and 50 additional controls employing customized Phenotype Microarray plates. The new plates will allow us to test 12 individuals per plate, using just 160,000 cells per individual. The proposed 100 samples, in addition to the 96 samples from the preliminary study, will give statistical power of 95.6% in a one- sided t-test at significance leve of 0.01. The second aim is to use our assay to evaluate the NADH production in the presence of tryptophan in fresh blood samples. As a first step, we plan to use 6 patients and 6 controls that we have already tested to replicate the results observed in lymphoblastoid cell lines in fresh white cells. This first stage will allow us to determine if we are able to observe the same low levels of NADH production in the presence of tryptophan in leukocytes as was observed in lymphoblasts. Next, we will test fresh blood samples from 12 patients with ASD and 12 controls. To reduce output variability, we will try to start the test at the same time for all the samples, coordinating the sampling of the 24 individuals. Finally, we will test 24 patient and 24 control blood samples from new individuals in a blinded fashion to establish the predictive power of this test in patients with ASD. For this last part of the experiment, we will test each sample as soon as it arrives at our laboratory. We will use a single row of 8 wells on the customized plates for each individual, covering the rest of the plate with aluminum foil, to preserve the other rows for future tests. If our preliminary findings in lymphoblasts are confirmed in leukocytes, we would be able to develop a reliable, easy to perform, inexpensive laboratory test which could provide a diagnosis in about 4-5 days. Such a test could represent the first biochemical screening test for ASDs. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a screening tool for Autism Spectrum Disorders (ASDs). In our preliminary study, we detected significantly lower levels of NADH generated in the presence of tryptophan in patients with ASD when compared to controls. We propose to utilize the same metabolic profiling approach using leukocytes from fresh blood samples to determine if ASDs can be quickly and reliably distinguished from controls.